**Abstract**

Earlier applications of photodynamic therapy (PDT) were accomplished by direct or intravenous injection of the photosensitizer, followed by preferential accumulation in cancerous tissues after systemic circulation. Nowadays, nanoparticles are used as carriers and delivery systems, which also facilitate combinations of PDT with other non-invasive technologies. PDT has expanded to disease types other than cancers. Nanoparticle-mediated target specific PDT can reduce the emergence of resistance, and has introduced chemotherapy combinations with PDT, and potential repurposing of chemotherapy drugs that are being used less because of resistance. The novel discoveries of inorganic and organic dye nanoconjugate photosensitizers discussed in this chapter have enhancement PDT efficacy. This review describes the type I and II mechanisms of PDT, some of the first- and second-generation photosensitizers in the market, and the roles played by nanomaterials across the PDT clinical translation value chain. It discusses nanoparticles as delivery systems for photosensitizers, smart stimulus-responsive, and disease-targeting nanoparticles, focusing on folate, glycanbased, pH, and external stimulus-responsive targeting. Well-known in anticancer applications, folate targeting is now debuting in antibacterial applications. Other targeting technologies are discussed. Nanoparticles applications as agents for combining PDT with other therapies are discussed. The World Health Organization has identified PDT as a promising new technology.

**Keywords:** antibacterial photodynamic therapy, photodynamic antimicrobial chemotherapy, nanoparticle-mediated photodynamic therapy, bacterial resistance, bacterial cell specificity, selectivity, drug carrier, drug delivery

### **1. Introduction**

From a clinical perspective, photodynamic therapy (PDT) may be defined as a treatment that involves the application of light energy in a disease-affected area where there is a sufficient concentration of the photosensitizer (PS). PDT destroys disease cells only upon activation by light, provided there is a sufficient oxygen concentration

in the disease. PSs are generally activated using laser light of a wavelength that is absorbed by the PS. They are nontoxic compounds that become toxic upon light activation. Clinical PDT is widely used against psoriasis; cancers of the skin, lung, brain, bladder, pancreas, bile duct, esophagus, and head and neck; as well as other diseases such as acne and age-related macular degeneration. Additionally, antimicrobial photodynamic therapy (aPDT) is used to treat bacterial, fungal, and viral infections. It has been established from several studies that there is an immune response to PDT that can further enhance its efficacy. From a mechanistic point of view, PDT involves the excitation of the PS to its singlet excited state upon absorption of light of a frequency that matches the absorption spectrum of the PS, followed by intersystem crossing to the triplet state, which is the state in which the PS either transfers energy to normal triplet state oxygen to produce excited singlet state oxygen or reacts with biomolecules, causing damage to cells. Singlet oxygen production is the most effective PDT pathway. It takes place under conditions of oxygenation and is referred to as type II mechanism. In contrast, the direct reaction of the excited PS with biomolecules, referred to as type I mechanism, predominates under conditions of hypoxia, because there is not sufficient oxygen for type II mechanism.

In the aPDT approach, absorbed light energy is used to achieve the bactericidal or bacteriostatic effect through these two critical molecular PS-mediated mechanisms. Here the type I mechanism involves much radical formation through hydrogen transfer from the PS directly to biomolecules, and the type II mechanism proceeds via oxygen photosensitization to produce a series of oxygen-based molecular species known as reactive oxygen species (ROS), which includes singlet oxygen, oxygen radicals, and hydroxide radicals and radical anions. All ROS react with biomolecules, causing irreversible damage [1]. The Jablonski diagram shown in **Figure 1** illustrates the two mechanisms. The irreversible chemical reactions that alter the functionality of biomolecules in bacterial cells and the extracellular polymeric substance (EPS) matrix [3], regardless of whether these biomolecules are cellular, EPS matrix components, or some other functional constituents of the biofilm [4], have been extensively studied. Many of these studies conclude that aPDT increases intracellular ROS and reduces the strength of the EPS matrix and the metabolic activity of the pathogen cells in the matrix [5].

Nanoparticles may be defined as ultra-small particulate materials with one of the dimensions of the particles up to 100 nanometers. Metallic nanoparticles like metal chalcogenide and silica nanocomposites have been reported. Self-assembled phospho-lipid porphysome vesicles [6] and phthalocyanine-based porphysome-like nanostructures [7] are very common PSs for PDT. Organo-inorganic nanomaterials comprise organic and inorganic nucleated heterocyclic aromatic organic compounds in self-assembled nanoparticle (NP) formations. Recently, metal organic frameworks (MOFs) have emerged, in which the linking organic molecules are PS molecules such as phthalocyanines and porphyrins [8, 9]. Typical applications of these PS molecules in PDT include antiviral, antibacterial, antifungal, anticancer, pest control, and environmental sanitization [2]. Several combination therapies, gene therapies, immunotherapies, and checkpoint blockade immunotherapies, in which these molecules are used as integral parts of PS nanoconjugate systems, have been widely reported. These nanoconjugates are widely reported in pharmaceutical formulation; controlled, stimulus-responsive, and slow drug release; enhancement of bioavailability; combination therapies; and enhancement of therapeutic efficacy, using a range of techniques such as nano-crystallization and self-assembly. Nanomaterials are found at every node of the therapeutic value chain and drug development pipeline, from

*Important Advances in Antibacterial Nanoparticle-Mediated Photodynamic Therapy DOI: http://dx.doi.org/10.5772/intechopen.113340*

#### **Figure 1.**

*Jablonski diagram to illustrate the aPDT type I and II mechanisms. Reproduced from Songca and Adjei [2] under the creative commons attribution license 4.0.*

basic drug research and development, through 2D and 3D evaluations in vitro, finally arriving at the preclinical studies in vivo, pharmaceutical formulation, applications in clinical trials, and drug administration in clinical therapy.

The challenge of incorporating PS molecules that are used for PDT and other drug molecules that are used as antibacterial chemotherapeutic agents, into innovative nanoconjugate systems, in designing them to act as carriers and delivery vehicles of the PS and drug molecules, and act as systems that respond to internal or external stimuli, once they are internalized into disease cells, is an important preoccupation of scientists in nanomaterial-mediated PDT. The purpose of incorporating PS molecules and antibacterial chemotherapeutic drug molecules into innovative nanoconjugate systems is to ensure their inertness and non-toxicity while in systemic circulation. The purpose of building internal and external stimuli responsiveness is to ensure that they are released only when the nanoconjugate is inside the target disease cell or site, when the stimuli of the internal environment of these cells or sites trigger their release, or when an external stimulus is applied (**Figure 2**).

Incorporating small molecules of antibacterial drugs as components of nanoconjugates presents many advantages in efficacy improvement. These include pharmacokinetic navigation of various physiological barriers and reduction of many of their side effects, including the development of bacterial resistance. Most of the self-assembly reactions used are conducted in aqueous media to form NPs composed of small, potent drug molecules. Most nanoconjugates are easy to fabricate; they can deliver high concentrations of their drug molecule cargo to the disease microenvironment and intracellular environment of the disease cells. Given the facile pharmacokinetic navigation of the systemic barriers by these drugs when capped or otherwise encapsulated in nanoconjugate form, they have great potential to reduce or eliminate their side effects because they are only released at the disease site and, in the most innovative designs, they are released only once they are inside the disease cells, and are not released inside normal host tissue cells.

#### **Figure 2.**

*Chemical structures of Foscan, Photofrin, Visudyne, Lutex, Pc4, Purlytin, HPPH, NPe6, Levulan, TLD1433, Hypocrellin a, and Hypocrellin B. The pharmaceutical companies are indicated in brackets.*

Most PSs used in PDT are organic molecular chromophores that are capable of transferring electromagnetic radiation energy to oxygen to form ROS in situ [10]. However, the new inorganic NP PSs that have been discovered are showing good versatility because in addition to being used for transport and delivery of the PSs, they can also act as PDT PSs, photothermal therapy, and magnetothermal therapy agents in combination therapies with PDT. For example, an inorganic NP PS consisting of Fe2O3, and CuS, which also acts as a PS and therefore, possesses photothermal and magnetothermal conversion, in addition to PDT capabilities, as reported by Curcio

#### *Important Advances in Antibacterial Nanoparticle-Mediated Photodynamic Therapy DOI: http://dx.doi.org/10.5772/intechopen.113340*

et al. [11]. The nanoconjugate demonstrated its capabilities in a tri-therapeutic combination involving photothermal hyperthermia therapy (PTT), PDT and magnetic hyperthermia therapy (MH), in which the iron oxide shell is responsible for MH, and the copper sulfide multi-core is responsible for PTT and PDT. In their review, Zhang et al. [12] identified carbon-based inorganic nanomaterials such as dots, fullerenes, nanotubes, graphene oxide semiconductor nanomaterials such as zirconium and titanium oxides, and defective nanomaterials such as oxides of ruthenium and zinc, as some of the inorganic NPs that generate ROS upon photo irradiation. Conjugation of these nanomaterials with the organic dye type of PS results in efficacious nanoconjugates in combination therapies. For example, the conjugation of copper sulfide NPs with chlorin-e6 produced a potent PDT-and-PTT combination agent because both the core and shell materials produce ROS [13].

Examples of organic PSs that have clinical approval include Foscan from Scotia [14], Visudyne from QLT [15], Lutex from Pharmacyclics [16], Pc4 from Case Western Reserve [17], Purlytin from Miravant [18], NPe6 from Nippon [19], HPPH from Roswell Park Cancer Institute [20, 21], Amino Laevulinic Acid from DUSA [22], Hypocrellin Photosensitizer SL052 from Canadian Quest PharmaTech [23], and TLD1433 from Theralase [24]. Examples of inorganic PSs include sulfides of molybdenum, zinc, copper, iron, silver, and bismuth [25]. Nanostructured MOFs [26] and metal complexes with organic ligands [23], on the other hand, may therefore be considered among the wide and increasing variety of organic–inorganic hybrid nanostructured PDT PSs [27–29].

#### **2. Purpose statement**

This paper presents the roles played by nanomaterials across the therapeutic value chain, from basic research to clinical applications, using examples from therapeutic technologies and their clinical applications against many bacterial and fungal diseases. The paper adopts an approach of considering therapeutic applications of nanotechnology in treating bacterial diseases and the nanomaterial-based therapeutic technologies applied in treating them, discussing the details of these applications and the technologies that define them. Using photosensitization type I and II mechanisms by which ROS are produced in the disease microenvironment, and the subsequent oxidative initiation of apoptosis and necrotic cell death, the purpose of this paper is pursued by discussing specific examples. The purpose of this approach is to provide the fundamental mechanistic basis of the technology and its combinations, an overview of its state-of-the-art from the current research and the historical viewpoint, the expansion of its scope, the enhancement of its efficacy and disease targeting, and the role of nanotechnology in these developments. This paper also aims to discuss potential areas of further research and innovation as indicated by gaps in the basic research and clinical translation literature.

#### **3. Nanoparticles as carrier and delivery systems for photosensitizers**

The use of NPs as carriers and delivery agents for PSs and other drugs has gained much attention [30, 31] and has demonstrated the enhancement of stability, solubility, administration, target delivery, specificity, selectivity, and toxicity reduction [32, 33]. Research on NPs as carriers of PSs has demonstrated that the enhancement

of PDT is due to the enhancement of PS drug delivery and cellular uptake and retention [34, 35]. Due to the ultra-small size of NPs, they have large surface-to-volume ratios [36]. This allows them to absorb large quantities of the PS on their surface [37], which promotes the target tissue and cellular uptake [38] once they reach the target site and cells. In addition, this PS drug delivery mechanism can also enhance selectivity for the disease site and cells over host tissue sites or cells. The absorption of PSs on the surface of NPs increases NP stability while in systemic circulation [39]. This severely limits undesirable side effects of both the NP and PS, such as toxicity in the absence of light. PS-capped NPs generally have improved solubility in hydroxylic media, thus enhancing the administration of the nanoconjugate [40]. The foregoing discussion describes the encapsulation of NPs by PSs. It may be illustrated using the example of encapsulation of magnetic NPs with heparin-pheophorbide-A, as reported by Li et al. [41], shown in **Figure 3**, in which the aminopropyl triethoxysilane functionalized iron oxide NPs are encapsulated with heparin–pheophorbide-A by conjugation of the functionalized NPs. The encapsulation of NPs by PSs is one of the most effective and therefore most widely reported strategies for using NPs as carrier and delivery systems of PSs for use in PDT.

The PS may be covalently linked or adsorbed onto the surface of the NP. For example, a near-infrared absorbing and disulfide functionalized bacteriochlorophylla-based PS was covalently anchored onto the surface of gold NPs for anticancer PDT, using gold surface–sulfide dative covalent bonding of the disulphide functional group of the PS [42]. The researchers found that in comparison to the free bacteriochlorophyll-a-based PS, the gold-PS nanoconjugate remained in systemic circulation for longer and showed increased tumor accumulation, cancer cell uptake and retention. This nanoconjugate is illustrated in **Figure 4**.

In this case, the linker is a functional group of the PS. The covalent anchoring of Rose Bengal on the surface of silica NPs, however, was accomplished by functionalizing the NP surface with amino groups followed by covalent linking of the PS, via the formation of amide covalent bonds between the carboxylic acid functional group of Rose Bengal and the amino groups of the silica capping shell [43]. This is illustrated in **Figure 5**.

The nanoconjugate inactivated gram-positive Methicillin-resistant *S. aureus* and *Staphylococcus epidermidis*, indicating that this method of PS conjugation has great potential for aPDT applications.

The encapsulation of PSs in the core of organic NPs such as liposomes and micelles has emerged as a powerful way of enhancing PS delivery [44]. This is a

#### **Figure 3.** *Heparin–pheophorbide-a conjugation of aminopropyltriethoxysilane functionalized nanoparticles.*

*Important Advances in Antibacterial Nanoparticle-Mediated Photodynamic Therapy DOI: http://dx.doi.org/10.5772/intechopen.113340*

#### **Figure 4.**

*Covalent binding of bacteriochlorophyll-a-based PS onto the surface of gold nanoparticles.*

#### **Figure 5.**

*Amide bond covalent binding via amino-functionalized silica nanoparticles.*

versatile approach because, while hydrophilic PSs are encapsulated in water-in-oil organic NPs, hydrophilic PSs are encapsulated in oil-in-water organic NPs [45, 46]. This is due to the respective structures of the oil-in-water and water-in-oil NPs. The constituent molecules of these organic NPs, known as micelles, are phospholipids, which self-assemble with the alignment of their hydrophilic heads and hydrophobic tails. Water-in-oil organic NPs align with hydrophilic heads in the interior of the NP, thus encapsulating hydrophilic PSs in an aqueous medium, while oil-in-water NPs align with hydrophobic tails in the interior of the NP, thus encapsulating hydrophobic PSs in an organic medium [47]. Unlike micelles, which have a single layer of phospholipids, liposomes have a double layer of phospholipids with an aqueous

#### **Figure 6.**

*Liposome with hydrophilic core hydrophobic bilayer, two partitions for hydrophilic and hydrophobic PSs respectively.*

core, encapsulating hydrophilic PSs and a hydrophobic phospholipid bilayer that can accommodate large quantities of hydrophobic PSs [48]. The structures of liposomes are illustrated in **Figure 6**.

To overcome the lipophilicity of porphyrins that limits their water solubility, protoporphyrin IX was conjugated with oleylamine to enhance its solubility in the liposomal bilayer [49]. **Figure 6b** and **7** illustrate the liposomal bilayer incorporation of the hydrophobic PS. The in vitro anticancer studies of the liposome incorporated PS showed that it significantly reduced the viability of HeLa and AGS cancer cell lines. Bilayer incorporation of the PS was also observed with temoporphyrin [50].

In contrast, the water-soluble PSs like Methylene Blue, Neutral Red, and Rose Bengal are encapsulated into the inner aqueous core of the liposome [51]. The liposomal encapsulation of these PSs, which was evaluated by gel filtration chromatography using Sephadex 100, is illustrated in **Figure 6a**.

Unlike the encapsulation of NPs discussed above, mesoporous NPs (**Figure 8**) have micro-pores that are large enough to absorb large quantities of PSs, permeating through their entire nanostructures. Mesoporous silica [53] and MOFs [54] are among the most widely studied mesoporous NPs. The advantage of using mesoporous silica NPs as PS carriers and delivery systems is that they are biocompatible and safe to use [55]. Mesoporous silica NPs are fabricated to assume several 3D structures, which enable loading and the control of NP release at the target site, and surface functionalization, depending on the synthetic methodology [56]. Dendritic mesoporous silica nanostructures have now emerged with highly porous nanostructures and high loading capacity due to their large pore sizes [57]. MOFs can absorb large quantities of PSs in their large pore sizes that can be hydrophobic or hydrophilic depending on the organic molecule linkers and the linked metal cations. As a result, they can absorb hydrophilic PSs in hydrophilic pore sites and hydrophobic PSs in hydrophobic pore sites [58]. Although most MOF pores tend to be hydrophobic due to the hydrophobic organic molecule linkers, the design and self-assembly of hydrophilic MOFs have been reported for the absorption of hydrophilic molecules such as glycol peptides [59]. Although, in theory, such MOFs can also be used to absorb hydrophilic PSs, to the best of our literature search, such research has yet to be reported.

A new type of MOF consisting of porphyrins or phthalocyanines as the organic linkers has been reported to absorb large quantities of oxygen, thus alleviating hypoxia in PDT and acting as PSs by ROS generation [60]. Furthermore, to ameliorate the tissue penetration challenge of normal light energy used in PDT, porphyrin-based MOFs

*Important Advances in Antibacterial Nanoparticle-Mediated Photodynamic Therapy DOI: http://dx.doi.org/10.5772/intechopen.113340*

**Figure 7.**

*Liposomal bilayer incorporation of the oleylamine conjugated protoporphyrin IX.*

#### **Figure 8.**

*Mesoporous silica nanoparticles and metal-organic-frameworks are highly porous nanomaterials. Copied from Zhang and Chang [52] under the creative common attribution license 4.0.*

have been reported, which absorb X-rays and transfer the energy to the porphyrin linkers for oxygen sensitization to generate ROS [61]. Other mesoporous nanomaterials developed for use in PDT include mesoporous carbon and titanium NPs. For example, oxygenated perfluoro hexane was loaded onto the mesoporous carbon NP channels for antibacterial applications in combining PTT and PDT [62]. Mesoporous titanium oxide NPs have been developed for overcoming drug resistance in a combination therapeutic approach involving disease targeting and drug delivery in PDT [63].
